Intestinal Barrier Physiology

INTESTINAL BARRIER PHYSIOLOGY

Genomic regulation of intestinal barrier functions with relation to lipid homeostasis.

Nutrition Metabolism and Genomics Group, Wageningen University, Dreijenplein 10, 6710 HB Wageningen, The Netherlands

The intestine as a gatekeeper plays a double-edge role in the uptake of dietary lipids. This is because absorption of dietary essential fatty acids and lipid micronutrients by the small intestine sustains human life, but at the same time, the efficient capacity of this organ to absorb our lipid-rich modern diet compromises and/or even harms people. Comprehensive detailed understanding of intestinal lipid homeostasis is therefore urgently needed. On a daily basis, the human small intestine metabolizes an average of 100 g of dietary fat, more than 90% of which is composed of triacylglycerols (TG). The absorption of lipids from the lumen is generally highly efficient, whereby approximately 4% of the ingested fat escapes into feces. Prior to absorption, TGs are hydrolyzed by gastric and pancreatic lipases to free fatty acids and 2-monoacylglycerols, both of which are taken up by enterocytes. We have comprehensively studied using microarrays and transgenic animals the effects of diets (rich in saturated and unsaturated fats) or pure fatty acids (in form of triglycerides) or of the PPARD ligand WY and of fasting on genome wide effects on intestinal barrier functions with a focus on lipid signaling and homeostasis.

1) We examined the effects of a high-fat diet on expression and function of cholesterol transporters in the small intestine in mice. Npc1l1, Abca1, Abcg5, and Abcg8 were all downregulated after 2, 4, and 8 wk on a cholesterol-free, high-fat diet. This fatty acid-induced downregulation of cholesterol transporters is LXRD

independent and associated with an induction of intestinal cholesterol biosynthesis as well as with a doubling of neutral fecal sterol loss. This shows induction of adaptive changes in small intestinal cholesterol metabolism during exposure to dietary fat.

2) The transcription factor PPARD functions as an important nutrient sensor in mediating the effects of dietary fatty acids on intestinal gene transcription by regulating many genes involved in uptake of fatty acids, intracellular metabolism and chylomicron formation. We first demonstrated that PPARD is highly expressed in villus cells of the duodenum and jejunum, coinciding with the primary anatomical location where fatty acids are digested, absorbed, and transported into the body as chylomicrons. Moreover, our genome wide analysis suggested that intestinal PPARD controls these processes, among others.

3) We also identified intestinal barrier genes that were PPARD-dependently regulated after acute activation by fatty acids including sets of transporters and phase I/II metabolic enzymes involved in a) fatty acid oxidation, b) cholesterol, glucose, and amino acid transport and metabolism, c) intestinal motility, and d) oxidative stress defense. This knowledge provides a better understanding of the impact dietary fat has on the barrier function of the gut, identifies PPARD as an important factor controlling this key function, and underscores the importance of PPARD for nutrient-mediated gene regulation in intestine.

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The desmosome-associated cellular prion protein contributes to the barrier function of

the intestinal epithelium

Constance SV Petita_{, Frédérick Barreau}b_{, Béatrice Riveau}a_{, Danielle Château}a_{, Monique Rousset}a_{, Caroline} Claira_{, Sophie Thenet}a,c

a_{Centre de Recherche des Cordeliers, Equipe 4, UMRS 872, 75006 Paris, France.,} 2_{INSERM U843, Hôpital Robert Debré, 75019 Paris,} France. c_{Ecole Pratique des Hautes Etudes, 75006 Paris, France.}

Non Digestible Oligosaccharides open tight junctions in the enterocyte cell line, Caco-2

Epithelial Sciences Research Group, School of Translational Medicine, University of Manchester, Manchester, UK

Introduction: non-digestible oligosaccharides (NDS) are part of the fibre component of the diet. In human intervention studies, NDS have been shown to impact positively on health in a several ways but the classical view of their actions is that they must be fermented by colonic bacteria to have any effects. NDS are not thought to interact directly with enterocytes.In this study we demonstrate that NDS open tight junctions in caco-2 cells and determine some of the molecular mechanisms involved. Methods: NDS including melibiose, lactulose and fruco-oligosaccharides were added to fully differentiated caco-2 cells growing on TranswellTM _filters. Transepithelial electrical resistance (TEER) was measured using an EVOM (World precision instruments). Activation of protein kinase C was performed using high throughput fluorescence based assay (Imagen Biosciences ltd, UK). Tight junction proteins were analysed using western blotting. Results: within 1 hour of exposure to NDS we observed dose dependent decreases in the TEER of caco-2 cells which only occurred when NDS were added apically. The TEER continued to decrease up to 2 hours post treatment. NDS had differing effects on TEER but the largest decrease of around 60% occurred within 2 hours of treatment with 100mM melibiose (p=0.02). By contrast, 50mM sucrose, a digestible saccharide, had no effect on caco-2 TEER.Within 10 minutes of adding NDS, we observed mobilisation of protein kinase C and the protein kinase C inhibitor GF 109203X prevented the NDS-induced reductions in TEER. The myosin light chain kinase inhibitor, ML-7, also prevented NDS induced reduction in TEER by around 40% (p=0.03). When cell extracts were analysed by western blotting, we observed an increase in the phosphorylation of myosin light chains in response to NDS.

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Effect of sodium butyrate on barrier function of HT-29/B6/GR-MR cells in conjunction

with down-regulation of claudin-2

Svenja Plögera_{, Theresa Bergann}a_{, Anja Fromm}b,c_{, Roland Bücker}c_{, Michael Fromm}b_{,Jörg D. Schulzke}a,c

a_{Department of Gastroenterology, Infectious Diseases and Rheumatology;} b_{Institute of Clinical Physiology;} c_{Department of General} Medicine, Campus Benjamin Franklin, Charité Berlin, Germany

Background and aims: The short chain fatty acid butyrate is known to have beneficial effects on colonocytes including preservation of barrier function or anti-inflammatory influences and plays also an important role in cell differentiation, apoptosis induction, and gene regulation by acting as a histone deacetylase (HDAC) inhibitor. Here, we aimed to characterize the barrier effect of butyrate via claudin-2.

Methods & results:HT-29/B6/GR-MR cells were incubated with 2 mM sodium butyrate. Transepithelial resistance increased to 176% after 24 h and to 246% after 48 h. Quantitative immunoblot analyses revealed after 24 h a downregulation of claudin-2 protein level to 23%. This result was confirmed in T84 and MDCK-C11 cells.Other barrier-relevant tight junction proteins like claudin-1, -4, -5, and -8 remained unchanged by butyrate. The HDAC inhibitor trichostatin A (TSA) had similar effects with a 39% decrease in claudin-2 expression, although not as intensive as butyrate. Real Time PCR analysis could confirm this result on mRNA level revealing 25fold degradation of claudin-2 mRNA to a fold-induction of 0.04 after sodium butyrate treatment. Neither changes in protein stability nor changes in mRNA stability did contribute to these changes. Electrophoretic mobility shift assays showed a reduced binding relation to a GATA binding-site within the claudin-2 promotor which points to transcriptional regulation by sodium butyrate being responsible for this effect.

Yersinia enterocolitica

induces barrier dysfunction in HT-29/B6 cells by causing discrete

“leaky regions” exhibiting tight junction alterations

Nina A Heringa_{, Jan F Richter}b_{, Susanne M Krug}b_{, Dorothee Günzel}b_{, Anja Fromm}a,b_{, Erwin Bohn}d_{, Rita} Rosenthalb_{, Roland Bücker}a_{, Michael Fromm}b_{, Hanno Troeger}c_{, Jörg D Schulzke}a,c

a_{Dept. of General Medicine;} b_{Inst. of Clinical Physiology;} c_{Dept. of Gastroenterology, Infectious Diseases and Rheumatology, Campus} Benjamin Franklin, Charité Berlin; d_{Inst. of Medical Microbiology and Hygiene, University Tübingen, Germany}

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Aerolysin, a pore-forming toxin of Aeromonas hydrophila, disturbs tight junction

integrity and wound healing

Roland Bückera_{, Susanne M Krug}b_{, Rita Rosenthal}b_{, Dorothee Günzel}b_{, Michael Fromm}b_{, Trinad Chakraborty}d_, Hans-Jörg Epplec_{,Jörg-Dieter Schulzke}a,c

a_{Dept. General Medicine;} b_{Inst. Clinical Physiology;} c_{Dept. Gastroenterology, Charité Berlin;} d_{Dept. Medical Microbiology and Virology,} University Giessen, Germany

Introduction. Aeromonas hydrophila causes gastroenteritis, septicaemia, and necrotising soft tissue wound infections. Although the pathogenesis of A. hydrophila and its enterotoxin/haemolysin aerolysin has been well described, the mechanisms of the epithelial barrier dysfunction and necrotisation are not completely understood.

Methods. Human colon HT-29/B6 monolayers were apically inoculated with isolates of A. hydrophila or by the secreted toxin aerolysin. Transepithelial resistances and macromolecule fluxes were determined in Ussing chambers. Paracellular and transcellular resistance were measured by two-path impedance spectroscopy (2PI). The subcellular distribution of tight junction (TJ) and cytoskeleton proteins was analyzed by confocal microscopy. The ability of wound healing was quantified in infected epithelial cells by induction of single cell lesions and measuring of recovery time.

Results. The main paracellular part of epithelial dysfunction had not been considered so far, since aerolysin has only been known to induce active chloride secretion. Here, we show that infection of HT-29/B6 cells more than doubled paracellular permeability to fluorescein and to 4-kDa-dextran. In parallel, 2PI measurements revealed a rapid and permanent decrease in paracellular resistance with either A. hydrophila

supernatants or aerolysin only.As structural correlate, TJ proteins were redistributed off the TJ by actomyosin contraction via intracellular calcium signalling.Moreover, we measured a defective epithelial wound closure through disturbance of cytoskeletal and TJ components. All effects could be prevented by pre-treatment with zinc, which inhibits aerolysin pore formation in the host cell membrane. The inhibition by zinc parallels the efficacy of zinc therapy of epithelial lesions and diarrhoea.

Absorption enhancers and jejunal uptake kinetics in vitro of methyl Į-D-glucoside

a_{Walther-Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians Universität München, Germany Nußbaumstrasse 26,} D-80336 München; b_{Research Center of Nutrition and Food Sciences, Biochemistry Unit, Technische Universität München, Germany}

So-called absorption enhancers (AEs) are proposed to increase small-intestinal absorption of poorly available drugs. While some of them inhibit the mucosal metabolism or active efflux of drugs, others increase drug permeability by impairing mucosal barrier function. Thus, this latter type of AE, e.g., deoxycholate (DOC), polyethyleneimine (PEI), decanoate (DEC), and ethylenediamminetetraacetate (EDTA) may impair sodium-dependent active transport proccesses, due to their different structures and properties not necessarily by the same mechanism, however. Therefore, these AEs were compared for their effect on the kinetic parameters of intestinal methyl Į-D-[14_{C]glucoside uptake. K}

M and Vmax were determined in vitro by means of 5-min incubations with everted rings of rat jejunum and by analysing the concentration-dependent tissue uptake according to Lineweaver-Burk (for results see Table).

control DOC PEI DEC EDTA

KM (mmol/l) 4.8 ± 0.9 6.9 ± 1.2* 6.6 ± 0.9* 4.5 ± 0.5 4.7 ± 0.5

Vmax (mmol/l/min) 11.3 ± 2.3 8.2 ± 1.0* 8.0 ± 1.2* 6.3 ± 0.4* 7.0 ± 0.8*

M ± SD, N = 6; * p < 0.05 (ANOVA)

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Comparison of tight junction barrier properties with expression of claudins along rat

intestine

Alexander G Markova_{, Anna Veshnyakova}b_{, Michael Fromm}c_{, Maren Amasheh}d_{, Salah Amasheh}c

a_{University of St. Petersburg, St. Petersburg, Russia;} b_{Leibniz Institute of Molecular Pharmacology, Berlin, Germany;} c_{Institute of Clinical} Physiology and d_{Department of Gastroenterology, Charite, Campus Benjamin Franklin, Berlin, Germany}

The tight junction (TJ) protein family of claudins is regarded to determine barrier properties in a wide variety of epithelia. Aim of the study was to compare epithelial barrier properties directly with the presence of TJ proteins in exactly defined segments along rat intestine.

Epithelial resistance (Repi_{) of duodenum, jejunum, ileum, and colon tissue preparations were measured} and discriminated from subepithelial resistance by impedance spectroscopy. In parallel, expression of TJ proteins was analyzed by Western blots and immune fluorescence confocal microscopy.

Repi _{was 40.8 ± 3.6, 28.2 ± 6.2, 22.8 ± 3.3, and 124.3 ± 4.3 Ÿ·cm}2 _{in duodenum, jejunum, ileum, and} colon, respectively. Thus, colon was characterized by higher Repi _{than more proximal segments. However, among} the small intestinal segments, Repi _{was highest in duodenum and lowest in ileum. Along the intestine, claudins -1,} -2, -3, -4, -5, -7, -8, and -12 were detected by Western blotting with different signal intensities. In accordance with Repi_{, colon showed strongest signals for tightening claudins -1, -3, -4, -5, and -8. The paracellular channel} claudin-2 showed strongest signals in ileum, and jejunum revealed strongest signals for claudin-12, but no signals for claudin-4 and -8. Duodenum showed weakest expression of pore-forming claudin-2, and strongest signals for tightening claudins -3, -4, -5, and -8, compared to jejunum and ileum.

CD97 attenuates colitis and strengthens intestinal adherens junctions by stabilization of

membrane-bound ȕ-catenin

Gabriela Austa_{, Susann Becker}a_{, Manja Wobus}a_{, Rick Schneider}a_{, Salah Amasheh}c_{, Doreen Sittig}a_{, Christiane} Kernera_{, Ronald Naumann}d_{, Jörg Hamann}e_{, Elke Wandel}ab

a_{Department of Surgery, Research Laboratories,} b_{Translational Centre for Regenerative Medicine, University of Leipzig,} c_{Institute of} Clinical Physiology, Charité, Berlin, d_{Transgenic Core Facility, MPI for Molecular Cell Biology and Genetics, Dresden,Germany;} e_{Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, The Netherlands}

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Functional regulation of tight junction proteins claudins 3 and 5 by shear stress and

hypoxia

Susanne Milatza_{, Andreas Zakrzewicz}b_{, Johannes Wolschner}a_{, Michael Fromm}a_{, Salah Amasheh}a

a_{Dept. of Clinical Physiology,} b_{Dept. of Physiology, Campus Benjamin Franklin, Charité Berlin, Germany}

Effects of PPARs on tight junction structure and function in Caco-2 cells

Epithelial Sciences Research Group, School of Translational Medicine, University of Manchester, Manchester, UK

Introduction: The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate the expression of several genes involved in diverse processes such as cellular differentiation, development and metabolism. Three PPAR isotypes have been identified: PPAR-Į, Ȗ and E/į. The subtypes are similar in structural homology yet have differential effects and expression patterns. In this study, we investigated the effects of PPAR agonists and antagonists on the development of the caco-2 cell tight junction.

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NEUROENDOCRINE REGULATION

Enteroendocrine cell function in intestinal inflammation

School of Translational Medicine, Stopford Building, University of Manchester, Manchester, M13 9PL

Background: Appetite is often impaired in patients with gastrointestinal inflammation, but the precise biological basis for this is extremely unclear. Gastrointestinal satiety signals are produced by enteroendocrine cells (EEC), and signal through endocrine and paracrine pathways to regulate food intake. Amongst other hormones, polypeptide YY (PYY) and glucagon like peptide-1 (GLP-1) possess satiety-inducing properties. Recent animal research has suggested that immune regulated upregulation of EEC activity plays a mechanistic role in the appetite and feeding disturbance observed during gut inflammation. This is a discovery that now requires clinical translation.

Methodology: Patients with small intestinal Crohn’s disease (CD) were studied, with active small and large bowel inflammation (SB and LB respectively), and age/sex matched controls. Gut peptide responses to a mixed nutrient test meal were studied using a multiplex-ELISA technique. Patient symptoms were assessed using a validated visual analogue score (VAS).

Results: CD patients displayed a significant reduction in appetite by VAS (p<0.0001). Total PYY was increased 2.15-fold in SB CD (p=0.04) but not LB CD. A significant correlation was observed between postprandial PYY responses and symptoms, specifically nausea and bloatedness (p=0.009 & 0.03 respectively). The postprandial rise in active GLP-1 in the SB CD group was more sustained when compared to controls (p=0.01). Ghrelin was 3-fold higher in the CD group compared to controls and showed reversed responsiveness by rising postprandially. This correlated with the Crohn’s disease activity index (p=0.02). Leptin showed no correlation to intestinal inflammation but showed significant correlation with the body mass index and a negative correlation with weight loss (p=0.0001 & p=0.01 respectively).

Intestinal epithelial stem/progenitor cells are controlled by mucosal afferent nerves

a_{Neuroscience and Physiology, Gothenburg University, Goteborg, Sweden.} b_{Department of Molecular Virology, Linkoping University,} Linkoping, Sweden.

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Mechanism of bradykinin B

receptor mediated action at submucosal ganglia from rat

distal colon

Martin Diener, Janine Avemary

Institute for Veterinary Physiology, Justus-Liebig-University Giessen, Germany

Bradykinin acts as inflammatory mediator in the gut. In the present study we characterized bradykinin-induced changes in the intracellular Ca2+ _{concentration ([Ca}2+_]

i) in whole mount submucosal preparations and membrane potential of cultured submucosal neurones from rat distal colon. Bradykinin (2·10–10 _{- 2·10}–7 _mol/l) evoked a concentration-dependent increase in [Ca2+_]

Influence of carbon monoxide on anion secretion in rat colon

Institute for Veterinary Physiology, Justus-Liebig-University Giessen, Germany

S40

Distribution of circadian rhythms along rat colonic crypts

a_{Department of Epithelial Physiology,} b_{Department of Neurohumoral Regulations, Institute of Physiology, Academy of Sciences of the Czech} Republic, Prague, Czech Republic.

Introduction: Organisms living in rhythmically changing environment possess timing machine based on molecular clocks in both the central nervous system and peripheral tissues including liver and alimentary tract where the clock genes exhibit rhythmic expression. The circadian clock regulates hundreds of functions including proliferation and intestinal digestion and absorption. We have shown recently in intestinal epithelium the rhythmic expression of Na(+)/H(+) antiporter Nhe3 and the cell cycle regulator Wee1, which are suggested to be clock-controlled genes. Moreover, we have demonstrated rhythmic expression of another ion transporter Dra

and g subunit of the sodium channel ENaC.

Aim: As these studies showed rhythms in scrapped mucosa from a large area of colonic epithelia, the goal of this study was therefore to elucidate whether there are differences in the distribution and phase shift of circadian clock and rhythms along the base-to-surface axis of colonic crypts.

Methods: Adult male Wistar rats were kept in light-dark regime LD 12:12. Sample collection was provided every 4 hours during 24 hours, colonic tissue was snap-frozen and histological sections were prepared using cryostat. Laser capture microdissection was used for specific tissue collection of crypt base and mouth, respectively. Total RNA was isolated and examined by quantitative real-time RT-PCR.

Results: We demonstrated the functional intestinal circadian clock (rhythmic expression of clock genes mRNA) in both crypt base and mouth with exactly the same phase of rhythm along the crypt axis. Furthermore we found rhythmic expression of the cell cycle regulator Wee1 mRNA in both crypt base and mouth and rhythmic expression ofgENaC and Nhe3 mRNA in apical part of crypts.

Conclusion: We determined phase-synchronized expression of clock genes and clock-controlled genes in basal vs. apical part of colonic crypts. The data demonstrate for the first time the functional synchronization of clocks in particular crypts.

Epithelial secretion of the porcine proximal colon is stimulated by submucous

neurotransmitters

Anja Mauksch, Gotthold Gäbel, Helga Pfannkuche Institute of Veterinary-Physiology, Leipzig University, Germany

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Melatonin effect on serotonin transporter in Caco-2 cells

Nyurky Matheus, Carmen Mendoza, Ruth Iceta, Eva Latorre, José E. Mesonero, Miguel .A Plaza, Pilar. Arruebo, Divina Murillo, Marta Castro, Laura Grasa, Ana Isabel Alcalde

Physiology. Department of Pharmacology and Physiology. Faculty of Veterinary Sciences. Zaragoza. Spain

MICRONUTRIENT TRANSPORT AND TOXICITY

Evidence that quercetin acts as an Fe

shuttle across MDCK cell plasma membranes

via GLUT1.

Evangelia Vlachodimitropouloua_{, Paul A Sharp}b_{, Richard J Naftalin}a

Departments of Physiologya _{and Nutrition & Dietetics}b_{, King’s College London, Waterloo Campus, Franklin Wilkins Building, London SE1} 9HN.

Introduction. The flavonone quercetin has several important properties; it is an intracellular antioxidant acting directly on oxidized substrates, it donates electrons to the transmembrane oxidoreductase DcytB, which transfers the electron to its extracellular domain where it can reduce extracellular oxidized substrates e.g. Fe3+ _to membrane permeable Fe2+_{(McKie et al. 2001, Science). Quercetin is both a high affinity inhibitor of glucose} transport via GLUT1, 3 and 4 and also binds at several sites within the central pore of GLUTs via which it is transported (Strobel et al. 2005, Biochem J; Cunningham et al. 2006, J Biol Chem). Unlike ascorbate, quercetin acts as a high affinity Fe2+ _{and Fe}3+ _{chelator, in both extracellular and intracellular solutions, and thereby} strongly attenuates Fenton reactions between Fe2+ _{and H}

2O2 that produce severely cytotoxic hydroxyl radicals (OH·_{). Since quercetin is both membrane permeable via GLUTs and a high affinity Fe}2+ _{chelator, we speculated} that it might act as a transmembrane Fe2+ _{shuttle via the GLUT1 transporter.}

Methods. To test this hypothesis we loaded MDCK cells overnight with FeSO4 (10ȝM), which werethen washed free of extracellular Fe2+ _{with PBS solutions containing the impermeant Fe}2+_{-specific chelator} desferrioxamine (DFO), 300ȝM. We then incubated the Fe2+_{-loaded cells with varying concentrations of} quercetin (0-100 ȝM), which was then removed by a further wash in quercetin-free solution containing both DFO and albumin (500 ȝM), which binds quercetin and thereby facilitates its extraction from cells. Cytosolic Fe2+ _{activity was determined by measuring Fe}2+_{-dependent luminescence following addition of 100 ȝM H}

2O2 and the OH·_{-sensitive luminescence probe LO12 to the cells (Wyman et al. 2008, FEBS Lett). Additionally, the} calibrated Fe2+ _{content of cell lysates, was measured either with the ferrozine reaction, or with the Fe}2+ -dependent LO12 luminescence, following addition of 100 ȝM H2O2.

Results. Fe2+ _{accumulation into cytosol showed a hyperbolic increase following 12 h incubation (half} maximal Fe2+ _{loading concentration = 22 ± 2 ȝM). Using a constant Fe}2+ _{loading concentration (10 ȝM) and} loading time (12 hours) quercetin incubation for periods of 2, 4 and 12 hours had a biphasic concentration effect on residual Fe2+ _{remaining within cytosol. In the range 0-0.1 ȝM quercetin, residual cytosolic Fe}2+ _fell proportionally to 25 % of the level without quercetin. Raising quercetin above 0.1 ȝM reduced Fe2+_extraction until with 50 ȝM quercetin there is zero Fe2+ _{extraction. Cytochalasin B (10 ȝM) or phloretin (10 ȝM) prevent} quercetin (0.1 ȝM) dependent Fe2+ _{extraction, indicating that the route of quercetin dependent Fe}2+ _{in the case of} MDCK cells is via GLUT1.

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Zinc transporters in the GI tract: expression and localization of members of the ZnT

subfamily

Chiara Murgia, Chiara Devirgiliis, Diana Bellovino, Guido Leoni, Giulia Ranaldi, Giuditta Perozzi. INRAN – National Research Institute on Food & Nutrition, Roma, Italy

Zinc is an essential micronutrient absorbed from the diet through the intestinal epithelium with the aid of specialized multi-span membrane transport proteins. Zn ions are necessary for virtually all cells and tissues in the body, being required for over 300 enzymatic activities, as well as for proper functioning of several regulatory and structural proteins. We have focused our research on the characterization of a subset of zinc transporters belonging to the SLC30A family and sharing several features, namely ZnT2, ZnT4 and ZnT8. These proteins share a quite high degree of structural and sequence homology, display different tissue-specificities, and they are all localized in the membrane of intracellular vesicles where Zn ions are accumulated for storage or required for specific functions. We have cloned ZnT4 as an inducible gene during intestinal differentiation. It was later reported that a point mutation in this gene represents the molecular basis of the lethal milk mouse syndrome, characterized by defective zinc secretion in the milk of lactating dams. We have studied the intestinal phenotype of ZnT4 lm/lm mice, proposing a compensatory zinc transport activity by the highly homologous transporter ZnT2. Our recent work is focused on the third member of this subfamily, which is mainly expressed in the endocrine pancreas. We have identified the functional form of ZnT8 as a homodimer resistant to dissociation by denaturing and reducing agents, which is properly assembled only in the presence of biological membranes. Evidence will be presented indicating that members of the ZnT 2-4-8 subfamily are able to associate in multimeric complexes. We are presently investigating through computer modelling and site-directed mutagenesis the potential role of specific aminoacid residues in affecting the stability of ZnT homodimers, with particular attention to cysteine residues, most likely involved in disulfide bridge formation.

Cell growth density affects the response to copper treatment of Caco-2 differentiated

monolayer

Manuela Natolia_{, Bruno Leoni}a_{, Igea D’Agnano}a_{, Rossella Brandi}b_{, Ivan Arisi}b_{, Armando Felsani}a_{,Maria Flavia} Zuccoa

a_{CNR, Institute of Neurobiology and Molecular Medicine.} b_{European Brain Research Institute, Rome, Italy.}

The human intestinal Caco-2 cell line has been extensively used as a model of the intestinal barrier. However, it is widely reported in literature that culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often lead to problems of reproducibility making difficult to compare results. We developed a new protocol in which Caco-2 cells were subcultured at 50% of confluence (Low Density, LD) instead of 90% of confluence, as usually reported in literature (High Density, HD). Using this new protocol, Caco-2 cells preserved a higher proliferation potential resulting in a cell population, which, reaching confluence was able to differentiate almost synchronously, forming a more homogeneous and perfectly polarized cell monolayer, as compared to that obtained with the HD cells. This comparison has been done by analyzing the gene expression and the structural characteristics of the 21-days differentiated monolayers by microarrays hybridization and by confocal microscopy. We investigated if these structural differences could also influence the effects of toxicants on 21-days-differentiated LD and HD cells. We analyzed the 2 hours-acute toxicity of CuCl2 in terms of actin depolymerization and MT2A (metallothionein 2A) and HSPA1A (heat shock protein 70) genes induction. Copper treatment resulted in different levels of actin depolymerization and gene expression induction in relationship with the culture protocol, the LD cells showing a more homogeneous and stronger response. Moreover, it is worthwhile to note, that in all these experiments, which have been repeated many times, the response of LD cells were always more reproducible and constant compared to the HD one. Our results suggest that different cell line maintenance protocols could influence a number of morphological and physiological properties of differentiated Caco-2 cells and these effects must be taken in account when these cells are used as intestinal model.

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Phytosterols impair vitamin D

uptake in Caco-2 cells

Aurélie Goncalves, Romain Bott, Patrick Borel, Emmanuelle Reboul UMR 1260 INRA-Université de Méditerranée, ERL 1025 INSERM, Marseille, France

Background: Adequate vitamin D status is necessary and beneficial for health, although deficiency and insufficiency are very common. Indeed, in addition to reducing the risk for bone disease, vitamin D likely plays a role in autoimmune diseases, cancer, heart disease, mortality, and cognitive function.

Phytosterols are commonly used in the diet to decrease cholesterol intestinal absorption in order to diminish hypercholesterolemia. As vitamin D displays a chemical structure close to the structure of cholesterol, we hypothesized that phytosterols may interact with vitamin D intestinal absorption as well.

Objective: Our aim was to evaluate the effect of cholesterol as well as the 3 main dietary phytosterols (E -sitosterol, stigmasterol and campesterol) on micellar cholecalciferol (vitamin D3) uptake in human intestinal cells.

Methods: We used differentiated Caco2-TC7 cell monolayers grown on filters. Cholecalciferol-rich mixed micelles, supplemented or not in sterol, were added to the cells for 60 min. A particular care was given to respect a ratio vitamin D/sterol close to the one observed in the diet. Cholecalciferol was then measured in rinsed scrapped cells and in basolateral medium (if any) by HPLC to evaluate its uptake.

Results: Cholesterol, E-sitosterol, stigmasterol and campesterol could significantly inhibit cholecalciferol uptake (up to 40%).

Influence of a reduced nitrogen diet on intestinal electrolyte transport in young goats

From monogastric animals and humans it is known that changes in dietary protein content can cause adaptive changes in electrolyte metabolism. A reduction of intestinal absorption of calcium (Ca) could be observed in monogastric animals and humans fed with a low protein diet.In ruminant feeding, one aim is to reduce environmental nitrogen (N) pollution by reducing dietary N intake. Since ruminants express recycling mechanisms very efficiently for saving N, it is expected that these recycling mechanisms protect ruminants in times of dietary N restriction. The aim of the present study was to determine whether there is an interaction between N and electrolyte homeostasis changing the intestinal absorption of electrolytes in N-restricted goats. Male white Saanen goats were fed either with an adequate (19.3%, n=7) or reduced N diet (6.7%, n=6). Plasma concentrations of phosphate (Pi) and total Ca were determined colorimetrically. Ionized Ca was detected by an ion-selective electrode. Epithelia from the proximal and mid jejunum were mounted into Ussing chambers. Radioactive tracers 45_{Ca or} 32_{P were used to calculate unidirectional Ca and P}

S48

Relationship between acetate and butyrate transport in the ruminal epithelium of sheep

a_{Dept. of Animal and Poultry Science, Univ. of Saskatchewan, Saskatoon, Canada;} b_{Inst. of Veterinary Physiology, Univ. of Leipzig,} Germany; c_{Inst. for Physiology, Pathophysiology and Biophysics, Univ. of Veterinary Medicine Vienna, Austria}

Apical uptake of acetate into ruminal epithelia of sheep comprises at least three components differing in their HCO3- dependence and NO3- sensitivity. The present study aimed to elucidate whether butyrate has related transport modes.

Materials and Methods: The transepithelial flux rates of [3_{H]-acetate and [}14_{C]-butyrate (10 mM each)} were determined in parallel in isolated ruminal epithelia of 17 sheep using Ussing chambers. In separate epithelia from the same sheep, apical uptakes of [3_{H]-acetate and [}14_{C]-butyrate were determined in parallel. The} chloride-free incubation buffer had a mucosal and serosal pH of 6.1 and 7.4, respectively. Bicarbonate-dependent (BD) flux or uptake was calculated as the difference between measurements in the presence (24 mM) and absence of HCO3-. Bicarbonate-independent (BI) flux or uptake was further divided into NO3--sensitive (NS) and NO3 -insensitive (NI) components based on flux or uptake inhibition by 40 mM NO3-.

Results: The total flux rate was higher for butyrate compared to acetate (2.70 ± 0.15 vs. 1.17 ± 0.10 μmol · cm-2 _{· h}-1_{; P < 0.05). This was linked to higher BD (1.14 ± 0.14 vs. 0.68 ± 0.10 μmol · cm}-2 _{· h}-1_{; P < 0.05) and} BI-NI components of flux rate (1.84 ± 0.15 vs. 0.40 ± 0.02 μmol · cm-2 _{· h}-1_{; P < 0.05). By contrast, the BI-NS} component of flux rate was smaller and negative for butyrate compared to acetate (-0.43 ± 0.19 vs. 0.084 ± 0.18 μmol · cm-2 _{· h}-1_{; P < 0.05). Total flux rates of butyrate and acetate (r = 0.91), as well as their BD (r = 0.69) and} BI-NI components (r = 0.61) were positively correlated (P < 0.01), while the BI-NS components were negatively correlated (r = -0.53; P < 0.05). Similar results were obtained for the uptakes of butyrate and acetate, except for an absent negative correlation between the BI-NS components (r = 0.34).

Changes in ion transport induced by the endogenous H

S donor, cysteine, across rat

distal colon

Ervice Pouokam, Martin Diener

Institute for Veterinary Physiology, Justus-Liebig-University Giessen, Germany

S50

Epithelial secretion of the porcine proximal colon is stimulated by submucous

neurotransmitters

Anja Mauksch, Gotthold Gäbel, Helga Pfannkuche Institute of Veterinary-Physiology, Leipzig University, Germany

Two novel colonic mineralocorticoid receptor cell models expressing functional

epithelial Na

channels

Theresa Berganna_{, Anja Fromm}b,c_{, Svenja Plöger}a_{, Sebastian Zeissig}a,d_{, Tim Ziera}e_{, Steffen A Borden}e_{, Michael} Frommc_{, Jörg D Schulzke}a,b

a_{Dept. of Gastroenterology, Infectious Diseases and Rheumatology;} b_{Dept. of General Medicine;} c_{Inst. of Clinical Physiology, Charité} Berlin, Germany; d_{Gastroenterology Division, Brigham & Women's Hospital, Boston, USA;} e_{Bayer Schering Pharma, Berlin, Germany}

S52

Functional characterization of IPEG-J2 Monolayer (as a model for native porcine

epithelium)

Andre Kacholdt, Mandy Bruch, Hanka Sanftleben, Annette Zeyner, Elmar Mohr University of Rostock, Germany

Background: In previous works native porcine epithelia was used to simulate the transport of proteins (GFP green fluorescent protein) from the gut into the blood. Because of the underlying biological unevenness of the tissue, transport rates of GFP were characterized by high inter- and intraindividual variability To minimize this effect, a cell culture (IPEG-J2) model was establish.

Methods: Cells growing in DMEM/Ham’s F12 with 15 % foetal calf serum and 36 mM sodium bicarbonate on a polyester membranes with 0.4 μm pores (corning costar 3801). TEER (Transepithelial electric resistance) values and the diffusion of ³H-manitol were measured. The transport rates of four essential ³H amino acids (Methionine M, Leucin L, Lysin K and Thyrosin Y) were determined. Also transport of small intact proteins (e.g. GFP) was investigated.

Results: The maximum of TEER and the minimum of diffusion of Mannitol (standard permeability coefficient Papp) have been reached on day eight after seeding (with a concentration of 600000 cells/mm). There was a transport of amino acids, glucose and sodium ions. In addition, first results showed transport of intact GFP, too. Glucose and sodium transport was detected in ussing-chamber by measuring electrical current. GFP transport was detected in the same system but measured by ELISA. Determining the kind of transport, further experiments are necessary. All described transports could also be detected on the native porcine epithelium.

Conclusions: The monolayer has the highest TEER values and the lowest Papp values on day eight. Transport of glucose, sodium and amino acids is similar to native tissues. Therefore, the cell culture model could be appropriate for transport investigations.

The project is supported by BMBF.

Primary rat enterocytes as a useful tool for the evaluation of drug uptake

a_{Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Germany} b_{Research Pharmacokinetics, Bayer Schering Pharma AG, Berlin, Germany}

S54

The intestine of marine teleosts: a model to study bicarbonate secretion.

Dipartimento Di Scienze della vita ”M.Malpighi”. Università di Messina. Viale Ferdinando Stagno d’Alcontres, 31-98166 S.Agata, Messina.

Marine teleosts, living in a hyperosmotic environment, undergo a continuous water loss. Dehydration is prevented by drinking seawater at high rate and by absorbing the fluid, desalinated at esophageal level, across the intestinal epithelium. In order to reduce the osmolality of the intestinal fluid and to minimize the intestinal absorption of excess calcium, this epithelium performs an active HCO3- secretion to precipitate the large amount of Ca++ _{present in the hypercalcemic external medium. In the present study, performed on the Dycentrarchus} labrax, we evaluated the possibility to study the mechanism of active HCO3- secretion in the isolated intestine mounted in Ussing chamber. Bicarbonate secretory fluxes were evaluated by the pH-stat method under short circuit conditions. We found that the tissue is able to secrete HCO3- at a rate of 2.1 ± 0.1 μeq•cm-2•h-1 (n=6) for at least three hours. This flux was dependent on the serosal Na+ _{and was inhibited by both luminal and serosal} DIDS. In order to determine the contribution of carbonic anhydrase in HCO3- secretion, experiments employing acetazolamide were performed. The role of transepithelial bicarbonate transport was tested in experiments performed in the absence of serosal HCO3- and CO2. The results obtained suggest that the main source of HCO3 -secreted is the extracellular HCO3- transported across the intestinal epithelium. The serosal uptake probably takes place by means of DIDS inhibitable Na+_{-dependent mechanism, while an apical Cl}-_-HCO

Fluorimetric analysis of copper transport mechanisms in B104 neuroblastoma cell

model: a contribution from cellular prion protein to copper supplying

Emanuela Ursoa_{, Antonia Rizzello}a_{, Raffaele Acierno}a_{, Maria Giulia Lionetto}a_{, Benedetto Salvato}b_{, Carlo} Storellia_{, Michele Maffia}a

a_{Department of Biological and Environmental Science and Technology, University of Salento, via Monteroni, 73100 Lecce, Italy;} b_{Department of Biology, University of Padova, via U. Bassi 58/B, 35121 Padova, Italy}

S56

Identification of novel inhibitors of intestinal phosphate transport: a potential approach

to controlling hyperphosphataemia in chronic renal failure

Johanne Marks, Robert J Unwin, Edward S Debnam London Epithelial Group, University College London, London, UK

Hyperphosphataemia is a serious consequence of chronic renal failure (CRF), leading to vascular calcification and increased cardiovascular morbidity and mortality. Targeting the kidney to reduce hyperphosphataemia in CRF is limited due to reduced renal function. Therefore, inhibiting intestinal phosphate absorption is a potential strategy for preventing phosphate overload in CRF. Currently, four phosphatonins have been identified: fibroblast growth factor 23 (FGF-23), matrix extracellular phosphoglycoprotein (MEPE), fibroblast growth factor 7 (FGF-7), and secreted frizzled related protein 4 (sFRP-4); all of which have been shown to have a potent inhibitory action on renal phosphate reabsorption. However, there is little information about the effects of phosphatonins on intestinal phosphate uptake. Our previous research has shown that MEPE acutely inhibits intestinal phosphate absorption independently of known regulators of phosphate homeostasis. The current study used an in vivo gut loop technique to show that two other phosphatonins have a similar inhibitory action on jejunal phosphate absorption. Thus, intravenous infusion of FGF-23 or sFRP-4 over a 3 hr period significantly decreased lumen to blood phosphate transfer to 67 ± 1.8 % (P<0.01) and 72 ± 4.1 % (P<0.05), respectively, compared with animals infused with vehicle only (90 ± 6 %). In contrast, FGF-7 infusion failed to evoke a significant change in phosphate absorption (80 ± 4.1 %).

These results demonstrate that three of the four known phosphatonins inhibit intestinal phosphate absorption in vivo. Studies are underway to investigate their potential to inhibit phosphate absorption in CRF as a means of controlling the associated hyperphosphataemia.

PiT transporters are regulated by age and diet

Sobiya P Nadarajaa_{, Johanne Marks}a_{, David Rosen}b_{, Robert J Unwin}a_{, Edward S. Debnam}a_.

a_{London Epithelial Group, University College London, London, UK;} b_{Acologix Inc, Hayward, CA, USA}

S58

Evidence that the colon may be important in phosphate homeostasis

a_{London Epithelial Group, University College London, London, UK;} b_{Acologix Inc, Hayward, CA, USA}

The importance of the small intestine (SI) for phosphate (Pi) absorption is widely accepted. There is some evidence for mRNA expression of Pi transporters in colonic epithelium (personal communication, Carsten Wagner), an intestinal region that has not hitherto been associated with Pi absorption. The purpose of our present study was to compare Pi absorption in the rat SI and colon, measured under both in vivo and in vitro conditions.

Copper involvement in gene expression, secretion and biological functions of TFF1

a_{Department of Pharmaceutical Sciences - Division of Biomedicine “Arturo Leone”,} b_{Department of Chemistry, University of Salerno,} Fisciano (SA), Italy

The Trefoil Factors Family includes the gastric peptides TFF1/(pS2), the spasmolytic peptide TFF2/(SP) and the intestinal trefoil factor TFF3/(ITF) which have an essential role in epithelial restitution within the gastrointestinal tract, where they are mainly expressed. We recently demonstrated that TFF1 is able to specifically bind copper ions through its acidic carboxy-terminal tail. Copper is an essential element for higher life forms, and plays a significant role in aerobic life as cofactor of enzymes involved in metabolically important red-ox reactions as oxidative phosphorylation, iron and oxygen transport, oxidative stress defences, collagen synthesis, etc. Our experimental evidence showed that copper binding favours the homodimerization of TFF1 protein, thus enhancing its motogenic activity. Work in progress is aimed at studying the involvement and influence of copper in tff1 geneexpression, protein secretion, and biological functions. Real Time PCR analyses showed that, in the human breast cancer cell line MCF-7, copper deficiency positively modulate tff1 mRNA, suggesting a regulatory role of copper in TFF1 expression. To this aim, we analyzed the regulatory role of the proximal upstream gene sequence in correlation with copper deficiency. In order to map possible copper responsive consensus elements, we analyzed the expression of the luciferase reporter gene driven by deletion constructs of the tff1 gene promoter. The human gastric cancer cell line AGS, transfected with the deletion constructs, showed that the upstream 5’ gene region -686/-472 is responsive to the variations of copper concentration. Namely, copper chelation with bathocuproine disulfonate (BCS) is able to stimulate an increase of gene transcription when the promoter contains the upstream region from -686 to +1, but it is not able to stimulate any increase when only -472 / +1 region is present. To investigate the involvement of copper in the TFF1 secretion process, we monitored the timing of TFF1 synthesis and secretion in a TFF1 inducible AGS clone (AGS-AC1). The results obtained show that copper overload delays the peptide secretion. Finally, preliminary atomic absorption analyses demonstrate that the uptake of copper in TFF1 hyper-expressing AGS-AC1 cells is faster than in non-induced cells. This evidence suggests that TFF1 levels may play a role in copper transport and homeostasis in this in vitro model.

S60

A protective role for zinc ions in the intestinal epithelium

INRAN - National Research Institute on Food and Nutrition, Roma, ITALY.

Zinc (Zn) is an essential micronutrient. Zn crosses biological membranes with the aid of specialized membrane transport proteins. Severe Zn deficiency is known to affect tight junction (TJ) integrity. Zn deficiency was often described in patients affect by chronic inflammatory intestinal syndromes such Crohn’s deases. In this study we investigated the hypothesis that subclinical Zn deficiencies, might lead to increased susceptibility of intestinal epithelial cells to toxic insults and/or inflammation. We investigated the effect of marginal Zn deprivation on the response of intestinal cells to the inflammatory cytokine TNFĮ. The same model was applied to study the effect of Zn status on the response of intestinal cells ochratoxin A (OTA), a harmful mycotoxin frequently contaminating a variety of foods. Zn-depleted or Zn-adequate Caco-2 cells were treated with TNFĮ or OTA.Marginal Zn deprivation makes intestinal cells sensitive to TNFĮ. While TJ integrity was unaffected after 8 h exposure to TNFĮ in Zn-adequate cells, Zn depletion caused a consistent increase of TJ permeability. Massive apoptosis was also observed. A gene expression analysis showed that a number of genes where modulated: the Inhibitor of Apoptosis cIAP2 up-regulation induced by TNFĮ was dependent by the integrity of Zn intracellular stores. The oncogene ErbB2 was downregulated by Zn depletion, especially in the presence of TNFĮ. Those results indicated that marginal Zn depletion caused intestinal cell response to TNFĮ to shift from survival to apoptosis with a mechanism that includes modulation of the expression of genes with key function in signal transduction pathway trigged by TNFĮ receptor. Intestinal cells are relatively resistant to high concentrations of OTA, Zn depletion of cells caused an increase of tight junction permeability in OTA treated cells, accompanied by increased apoptosis. A complex perturbation of Zn homeostasis was also demonstrated. Our results support the evidences that Zn has an anti-apoptotic function in intestinal cells and it is able to protect intestinal cells from OTA injury and inflammatory mediators. This suggests that imbalance in Zn transport and homeostasis, and/or Zn nutritional deficiencies could increase intestinal epithelial susceptibility to toxic insults.

Comparison of butyrate uptake by two non-transformed intestinal epithelial cell lines

Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, Porto, Portugal

The aim of this study was to characterize the apical uptake of BT by two distinct non-transformed intestinal epithelial cell lines, namely the rat small intestinal epithelial cell line (IEC-6 cells) and the human fetal epithelial colon cell line (FHC cells). Apical uptake of 14_{C-BT (20 μM) by FHC cells was: (1) time-dependent,} (2) concentration-dependent, with a Km of 1.22±0.45 mM and a Vmax of 109±17 μmol/mg prot/3 min, (3) pH-independent, (4) Na+_{- and Cl}-_{-independent, (5) energy-independent, (6) not inhibited by several BT structural} analogues (propionate, D-ketobutyrate and lactate), and (7) insensitive to monocarboxylate transport (MCT1) inhibitors (NPPB and pCMB). These characteristics suggest a lack of involvement of MCT1-mediated transport in the uptake of 14_{C-BT. Apical uptake of} 14_{C-BT (10 μM) by IEC-6 cells was: (1) time-dependent, (2)} concentration-dependent, with a Km of 4.01±1.1 mM and a Vmax of 70±14 μmol/mg prot/3 min, (3) pH-dependent, being strongly stimulated at an acidic pH, (4) Na+_{- and Cl}-_{-dependent, (5) energy-dependent, (6)} inhibited by several BT structural analogues (propionate, D-ketobutyrate, acetate, pyruvate and lactate), and (7) sensitive to MCT1 inhibitors (NPPB and pCMB). These characteristics are compatible with the involvement of MCT1, and possibly also of the sodium-dependent monocarboxylate transporter 1 (SMCT1), in 14_{C-BT uptake.} In conclusion, apical uptake of 14_{C-BT by FHC and IEC-6 cells shows rather distinct characteristics. Moreover,} comparison of the absolute amount of 14_{C-BT taken up by FHC and IEC-6 shows that the former cell line has a} very limited capacity to take up BT.

S62

The effect of folate status on the uptake of physiologically relevant compounds by

Caco-2 cells

Sandra Tavaresa_{, Joana Sousa}a_{, Pedro Gonçalves}a_{, João R Araújo}a_{, M João Pinho}b_{, Fátima Martel}a

a_{Department of Biochemistry (U38-FCT),} b_{Institute of Pharmacology and Therapeutics (U38-FCT), Faculty of Medicine, University of Porto,} Porto, Portugal

The aim of this work was to investigate the effect of folate status on the uptake of several physiologically relevant substances by Caco-2 cells. For this, Caco-2 cells cultured in high-folate conditions (HF) were compared with cells cultured in low-folate conditions (LF). Growth rates of HF and LF Caco-2 cells were similar. However, proliferation rate of LF cells was significantly greater than that of HF cells during the first two days of culture and slightly smaller thereafter, viability of LF cells was greater than that of HF cells, and apoptosis index was similar in both cell cultures. We verified that in LF cells, comparatively to HF cells: (1) uptake of 3_{H-folic acid was upregulated, via a significant increase in the V}

max of uptake; (2) uptake of 3 H-deoxy-glucose, 3_{H-O-methyl-glucose and} 3_{H-1-methyl-4-phenylpyridinium (MPP}+_{) was downregulated, via a decrease} in the Vmax of uptake; additionally, a reduction in Km was observed for 3H-O-methyl-glucose; (3) uptake of 3 H-5-hydroxytryptamine and 14_{C-butyrate was not changed; (4) the steady-state mRNA levels of the folic acid} transporters RFC, PCFT and FRĮ, of the organic cation transporter OCT1, of the glucose transporter GLUT2 and of the butyrate transporter MCT1 were significantly decreased.

In conclusion, folate deficiency produces substrate-specific changes in the uptake of bioactive compounds by Caco-2 cells (folic acid uptake is upregulated, glucose and organic cation (MPP+_{) uptake is downregulated,} and butyrate and 5-hydroxytryptamine uptake are not changed). Moreover, these changes are associated with alterations in the steady-state mRNA levels of specific transporters for these compounds.

REGULATION OF NUTRIENT TRANSPORT

The regulation of intestinal iron transport by leptin

King’s College London, Nutritional Sciences Division, London UK.

Obesity is associated with an increase in the production and release of a cocktail of pro-inflammatory cytokines from adipose tissue. In many obese subjects this low-grade inflammatory response leads to poor iron status or anaemia. Iron homeostasis is controlled by the liver-expressed iron regulatory peptide hepcidin. In anaemia of chronic inflammation, cytokine release leads to increased production of hepcidin, which in turn restricts intestinal iron absorption and iron release from reticulo-endothelial macrophages. Here we have focused on the role of leptin in controlling iron homeostasis. Leptin induces hepcidin expression (Chung et al J Nutr 2007 137:2366–70) and we have therefore investigated whether the leptin-hepcidin pathway influences intestinal iron absorption. In addition we explored the possibility that leptin might act directly on intestinal epithelial cells to regulate iron transport.

Studies were performed using Caco-2 cells grown on Transwell inserts and either co-cultured with HuH7 hepatoma cells pre-stimulated with leptin to produce hepcidin, or mono-cultured in the presence or absence of human recombinant leptin. Iron transporter (DMT1 and FPN) expression was determined by western blotting. The effects of leptin on intestinal iron transport were measured using 59_{Fe. Statistical analyses were performed} using Student’s unpaired t-test or 1-way ANOVA.

S64

Impaired GLUT2 trafficking in enterocytes of human obese subjects and fat-fed mice.

Centre de Recherche des Cordeliers, a_{UMRS872Team 9,} b_{UMRS872Team7,} c_{Pitié Salpétière Nutrition and obesity department and} d_Surgery department and e_{Pathology department of Hotel Dieu Hospitals, Paris, France}

Transport and metabolism revisited: a novel tryptophan transporter in cells expressing

indoleamine and 2, 3 dioxygenase.

Richard Boyd

Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road, Oxford OX1 3QX United Kingdom

S66

Functional characterisation of human Proton Amino Acid Transporter 4 (hPAT4)

expressed in

Xenopus

oocytes

Samyuktha Muralidharan Pillai, David Meredith

School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, UK.

Transport of some disaccharides in rat and human small intestine. The

enzyme-transport complex really exists

Sergey T Metelsky

Institute of general pathology and pathophysiology RAMS, Moscow, Russia

Background. A. Ugolev (Ugolev, 1989) suggested that membrane hydrolysis and transport of some oligomers of nutrients are performed by enzyme-transport complexes (ETC). Such complex would work effectively if the released monomers are transferred to the transporter input without appearing in the surrounding solution.

Aim. To compare the properties of enzyme-transport complexes for the disaccharide maltose (glucose + glucose) with those of Na+_{-dependent transporter for glucose in rats and humans.}

Methods. In isolated samples of the rat (n=85) and human (n=19) small intestine we recorded the pairs of successive responses of the short-circuit current (SCC) to the addition of maltose and glucose tothe washing solution. Maltose and glucose were added in the ratio 1:2 (by concentration). The magnitude of the SCC response on sugar characterizes the rate of Na+_{-dependent transport of this molecule.}

Results. In rats, SCC responses to 5 mM maltose and 10 mM glucose under a wide range of experimental conditions were practically equal. The correlation coefficient was equal to 0.992, the linear regression equation Y = 0.1 + 1.05 * X. Disaccharide cellobiose (glucose + glucose, bond by another type of link than that in maltose) non-hydrolyzed in rat intestine, did not stimulate the SCC. The stimulating effect of 10 mM of poorly hydrolyzed in adult rats lactose (glucose + galactose) was much smaller than effects of simultaneous addition of 10 mM glucose + 10 mM galactose or 20 mM glucose to mucosal solution. Comparingthe dynamics of sugars diffusion through unstirred layer to brush border membrane with the dynamics of the SCC response to the addition of maltose and glucose one can conclude that glucose derived from maltose molecules apparently did not appearin the surrounding solution. Data obtained on the human small intestine indicate that the ratio of the SCC responses on 5 mM maltose and 10 mM glucose may be either less or more than 1.0. Correlation coefficient was equal to 0.551.

S68

Chloroplast thylakoid membranes retard intestinal uptake of glucose

a_{Dept of Experimental Medical Science, BMC,} b_{Dept of Biochemistry, Chemical Centre,} c_{Dept of Biology, Lund University, Lund, Sweden}

Transport studies in human intestinal Caco-2 cells under optimized culture conditions

using serum-free medium.

Yula Sambuya_{, Simonetta Ferruzza}a_{, Carlotta Rossi}a_{, Mirjam Spansier}b_{, Manuela Natoli}c_{, Maria Laura Scarino}a_.

a_{National Research Institute on Food and Nutrition (INRAN) - Rome, Italy,} b_{Merck Research Laboratories - Oss, The Netherlands,} c _Institute of Neurobiology and Molecular Medicine (INMM), Rome, Italy

The human intestinal Caco-2 cell line represents the best available model for studies of intestinal transport, metabolism and toxicity, and has been extensively used for over two decades in laboratories all over the world. However, several reports in the literature point to great heterogeneity and variability in the expression of differentiated functions in this experimental model that can largely be ascribed to differences in culture procedures. Addition of foetal bovine serum (FBS) in the culture medium represents a source of variability in the performance of the Caco-2 cell line. We have been investigating different protocols to achieve optimal differentiation of Caco-2 cells in the absence of the serum supplement. In particular, permeability properties and transport by the apical carriers Pgp and PepT1 have been investigated in cells differentiated for up to 21 days on polycarbonate filter inserts in medium supplemented with different serum substitutes in the basolateral medium. These included insulin, transferrin and selenium (ITS) supplemented with a lipid mixture made of oleate, palmitate and cholesterol (ITS/Lipids) or MITO+© _{Serum Extender, a commercial mixture of hormones and} growth factors, with or without addition of the lipid mixture. The active transports of cephalexin (by PepT1) and of digoxin (by Pgp) in cells differentiated in the presence of MITO+ were expressed to the same or even higher levels than those of control cells differentiated in the presence of serum. Permeability to mannitol was unaffected in all conditions of serum-free medium, indicating functional closure of the tight junctions, although the presence of the MITO+ supplement resulted in lower TEER values, in line with the lower electrical resistance of the small intestinal epithelium compared to the colon.

Several laboratories still utilize an impermeable plastic substrate instead of permeable filter supports to differentiate Caco-2 cells, especially when accessibility to the basolateral membrane is not required (i.e. for gene expression, intracellular metabolism, apical activities or uptake studies). We therefore investigated the effects of plastic or filter substrates on the differentiation properties of Caco-2 cells. Our results indicate that gene expression, enzyme activities, morphology and protein localization are strongly affected by the nature of the substrate and that an impermeable substrate such as plastic does not allow full and homogeneous differentiation of the cell monolayer.

S70

The efficiency of coupling between hydrolysis of oligosaccharides and absorption of

glucose in the small intestine

in vivo

Andrei Gruzdkova_{, Liudmila Gromova}a_{, Nadezhda Grefner}b_{, Yan Komissarchik}b

a_{I.P.Pavlov Institute of Physiology, RAS,} b_{Institute of Cytology, RAS, St.-Petersburg, Russia}

The rates of maltose and maltotriose hydrolysis, and glucose (free one, and released from oligosaccharides) and water absorption in isolated loop of the rat small intestine were determined during a sequential perfusion of the loop (~18 cm) with solutions of the substrates in equivalent concentrations under conditions of chronic experiments. By means of immunofluorescent analysis, the distribution of glucose transporters SGLT1 and GLUT2 in the enterocytes was investigated at various levels of intestinal villi, and at low (25 mM) and high (200 mM) glucose loads.The coupling coefficient (a ratio of the rate of released glucose absorption to the rate of oligosaccharide hydrolysis) proved to be the highest (0.783) under perfusion of the loop at the rate of 0.26 ml per min with maltotriose solution (66.6 mM). In all experiments, rates of free glucose (G-glucose) absorption did not differ significantly from the rates of M- and MT-glucose absorption from equivalent maltose and maltotriose solutions, respectively. Rates of water absorption from the loop were significantly higher under perfusion of the loop with maltose (100 mM), and maltotriose (66.6 mM) solutions as compared with those under perfusion with equivalent glucose solutions. The distribution of SGLT1 and GLUT2 in the enterocytes of intestinal villi was different at low and high glucose loads. The transporters were visualized in the villus upper one-third at low glucose load and in total length of the villus at high glucose load. A mathematical model has been developed which takes into account the geometric peculiarities of the small intestinal absorptive surface, and A.M. Ugolev’s hypothesis that the affinity of glucose, just released from oligosaccharides, is higher than that of free glucose. Results of simulation are in a good accordance with the corresponding experimental data. The model predicts that under physiological conditions a transfer of the substrates across the pre-epithelial layer via solvent drag plays a minor role as compared with their diffusion across this layer.

Characterization of the human glucose cotransporter GLUT12

a_{Department of Biochemistry, Austral University of Chile, Valdivia, Chile.} b_{Department of Physiology, Toxicology and Nutrition, University} of Navarra, Pamplona 31008, Spain.

The facilitative glucose transporters GLUT/SLC2A are integral membrane proteins widely distributed in mammalian cells. Until now fourteen isoforms have been identified, which have been subdivided into three different classes (I, II and III) based on the amino acid sequences. In the present work, we have cloned GLUT12 (class III) from the mammary cancer cell line MCF7 and studied its kinetic characteristics and sensitivity for classical GLUT transporters inhibitors, still unknown. GLUT12 was expressed in Xenopus laevis oocytes and kinetic parameters were obtained using uptake assays. Selectivity for inhibitors was studied on transfected MDCK cells. The studies were performed in sodium buffer at pH 7,5. 3-O-methyl-D-glucose showed a K0.5 of 5mM and Vmax of 24 pmoles/oocyte/min. In the absence of Na+ the sugar uptake was decreased by 50%, which suggested the possibility of cotransport activity for GLUT12. Preliminary electrophysiological experiments in oocytes expressing the transporter demonstrated the presence of Na+ _{currents at saturating sugar concentrations.} The inhibition studies performed in transfected cells showed sensitivity of GLUT12 to cytochalasin B and inhibitors of GLUT1 like genistein, quercetin and tyrphostin A47. Based on these results, we suggest that GLUT12 could function as a cotransporter like the H+_{/myoinositol transporter, which also belongs to class III.} Work in progress will allow us to characterize more deeply this transporter.

S72

The absorption of glucose and the localization of SGLT1 and GLUT2 in ɋɚɫɨ-2 cells at

different glucose loads

Nadezhda Grefnera _{Liudmila Gromova}b_{, Andrei Gruzdkov}b_{, Yan Komissarchik}a

a_{Institute of Cytology, RAS,} b_{I.P.Pavlov Institute of Physiology, RAS, St.-Petersburg, Russia}

Leptin and insulin control the activity and expression of intestinal hexose transporter

SLC2A7/GLUT7

Sakar Yassine, Nazaret Corinne, Bado André, Ducroc Robert Physiologie et Neuroendocrinologie Digestive, Inserm U773, CRB3, Paris, France

GLUT7 (SCL2A7) is a new facilitated hexose transporter that has been primarily identified in the intestine (Li et al 2004). This transporter exhibits high affinity for fructose and glucose in vitro. The regulation of expression/activity of this GLUT7 transporter is currently unknown. Here we address this question by studying the effects of leptin and insulin, two distinct regulators of GLUTs activity.

Methods: Wistar rats and C57BL/6J mice were used after a 16-hour food deprivation period. Under anaesthesia, jejunal loops were isolated in situ and were randomly pre-treated 3 min with intraluminal 1 nM leptin. Thereafter the loops were challenged with either 30 mM fructose or 30 mM glucose. Insulin (1 UI/kg) was given i.p 3 min after sugars. After 5 min or 3 hrs, brush-border membranes (BBM) or total RNA were extracted from mucosa scrapings for western blot analysis of GLUT7 protein (antibody ABIN154302) or quantitative PCR of GLUT7 mRNA levels, respectively.

Results: In the isolated rat jejunal loops, glucose or fructose added into the lumen significantly increased the GLUT7 protein in BBM by 1.5 fold and 1.25 respectively compared to control. Luminal leptin alone significantly increased and i.p insulin significantly decreased the GLUT7 abundance in BBM. A combination of leptin with glucose or fructose gave GLUT7 levels of 2.0- and 1.9 fold higher amounts than those observed in controls, (P<0.05). Finally, in the mice in vivo oral leptin significantly increased (+23%) the GLUT7mRNA levels compared to control.

S74

‘Junk-food’ diet-induced obesity has different effects on glucose transporters in the

kidney and jejunal brush border membrane

Havovi Chichger, Marie Houdmont, Johanne Marks, Scott Wildman, Edward S Debnama_{, Robert J Unwin.}

London Epithelial Group, University College London, London, UK; Royal Veterinary College, London, UK

The epidemic of obesity that has developed in the last 20 years is thought to be largely diet-related, and it is associated with a range of clinical disorders, including hypertension and type II diabetes. Diabetic hyperglycaemia in rats induces expression of GLUT2 at the proximal tubule (PT) brush border membrane (BBM